Genital herpes simplex virus-2 (HSV-2) infection is one of the most common sexually transmitted infections (STI) worldwide. New strategies to address the growing HSV-2 epidemic are needed. HSV-2, like many STI, disproportionately affects women and establishes life-long recurring infections by periodic reactivation of latent virus. HSV-2 genital lesions are associated with enhanced HIV susceptibility and infection. HSV-2 symptoms can be treated with antivirals, but there are no available preventative vaccines. To better understand the host response to primary HSV-2 infection we utilized our human 3-D organotypic vaginal epithelial cell (VEC) model that more accurately recapitulates in vivo human vaginal tissue (e.g., barrier properties and function). Using a PCR array, we evaluated the expression of 84 genes associated with inflammation and signaling following HSV-2 infection. We identified the novel IL-1 family member, IL-36?, as it was significantly upregulated (~20-fold) by HSV-2 infection. Our central hypothesis is that IL-36? functions as a key regulator of mucosal inflammation in the female reproductive tract (FRT). Since the discovery of IL-36? (also referred to as IL-1F9) over a decade ago, there are only 15 publications on this cytokine and there is little known regarding the physiological function of this molecule. IL-36? is secreted by keratinocytes as well as at the respiratory and GI tracts, however expression of this cytokine has not been shown in the FRT. Notably, IL- 36? has been shown to play a role in chronic inflammatory disorders of the skin (e.g., psoriasis) by regulating skin inflammation. In psoriatic tissue, IL-36? was shown to induce matrix metalloproteases and antimicrobial peptides, critical elements of the mucosal barrier function in the FRT. Barrier disruption and sustained inflammation in the vagina can predispose to STI acquisition, however the role of IL-36? in these processes during HSV-2 infection is unclear. The long-term goal is to gain a fundamental understanding of the role of IL- 36? with regard to function, regulation and promotion of FRT mucosal inflammation, barrier function and HSV-2 pathogenesis. The rationale for the proposed research is that once it is known how IL-36? is functioning in the context of HSV-2 infection in vitro (Aim 1) we may be able to further understand how this novel cytokine contributes to the disruption or maintenance of mucosal homeostasis in the FRT (Aim 2) and its role in HSV-2 pathogenesis in vivo (Aim 3). To achieve these aims we will employ our human 3-D VEC model (Aims 1, 2) in the proposed in vitro studies. To further investigate the role of IL-36? in HSV-2 disease we will use a genital HSV-2 mouse model (Aim 3). The proposed work capitalizes on our novel findings with IL-36? employing our 3-D human VEC model. At the completion of this project, we expect that the proposed work will provide essential information to expand our fundamental understanding of IL-36? induction/regulation at the vaginal mucosa. We also expect to begin to elucidate the physiological and biological functions of IL-36? as a critical component of the inflammatory signature associated with mucosal inflammation and HSV-2 pathogenesis.